9种WADA禁用生长激素释放肽和生长激素促分泌素的HPLC-MS/MS检测及尿中稳定性研究
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摘要
目的:选取世界反兴奋剂机构(WADA)禁用的7种生长激素释放肽(GHRP-1,GHRP-2,GHRP-4,GHRP-5,GHRP-6,Hexarelin和Alexamorelin)和2种生长激素促分泌素(Anamorelin和Ipamorelin)为研究对象,建立人尿中的HPLC-MS/MS检测方法,进行稳定性实验。方法:使用Oasis?WCX固相提取小柱(1cc,30mg)对尿样进行化学前处理,尿样加入内标后离心,取1m L加入小柱,依次用5%NH4OH和20%CH3CN清洗后,用含2%甲酸的水/乙腈(1/3)洗脱液洗脱,35℃氮气流下吹干,复溶后进样于LC-MS/MS。使用1290/6470液相色谱质谱仪和Zorbax 300SB-C18色谱柱进行定性分析,流动相采用含0.2%甲酸的水/乙腈体系。结果:检测限根据不同物质分布在0.01~0.5 ng/m L(S/N>3)。回收率分布在40%~76%之间,日内精密度和日间精密度均小于15%,尿中基质无明显干扰。室温,冷藏条件储存和反复冻融对Anamorelin、GHRP-2、GHRP-4和GHRP-5有明显影响。结论:建立了尿中9种生长激素释放肽和生长激素促分泌素的HPLC-MS/MS检测方法,方法简单,特异性、灵敏度均可满足WADA实验室国际标准和技术文件要求。现已应用于我实验室的常规检测。尿样在传送、检测及实验室长期保存过程中应尽量避免反复冻融。
Objective To introduce a practical high performance liquid chromatography-tandem massspectrometry(HPLC-MS/MS)method for the detection of seven growth hormone-releasing peptides(GHRPs)including GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, Hexarelin and Alexamorelin andtwo growth hormone secretagogues(GHS)including anamorelin and ipamorelin, and study the stability ofthese nine substances in the human urine. Method The urine samples were purified and extracted bya solid phase extraction procedure using Oasis?WCX column. The urine was first centrifuged and tak-en out 1 m L into a small column, cleaned by 5% NH4 OH and 20% CH3 CN respectively, eluated usingthe mixture of water and acetomitrile(1/3)with 2% formic acid, blow-dried in the nitrogen at 35℃ andfinally redissolved to be injected into the LC-MS/MS. Result The limits of detection were between0.01~0.5 ng/m L accordingly. The spiked recoveries at the low concentration(1 ng/m L), medium concentration(2 ng/m L)and high concentration(10 ng/m L)ranged between 40% and 76%. The intra-and inter-day precisions of the target substances at these three concentrations were all less than 15%. The indoortemperature, refrigeration condition and multigelation were observed to have significant impact on theanamorelin, GHRP-2,GHRP-4 and GHRP-5. Conclusion The method established in this study is sim-ple, and its specificity and sensitivity meets the international standard and technical documents for labo-ratories set up by the Wworld Anti-Doping Agency. It has been applied in our routine work. Multigela-tion should be avoided in the transport, detection and long-term laboratory storage of urine samples.
Objective To introduce a practical high performance liquid chromatography-tandem massspectrometry(HPLC-MS/MS)method for the detection of seven growth hormone-releasing peptides(GHRPs)including GHRP-1, GHRP-2, GHRP-4, GHRP-5, GHRP-6, Hexarelin and Alexamorelin andtwo growth hormone secretagogues(GHS)including anamorelin and ipamorelin, and study the stability ofthese nine substances in the human urine. Method The urine samples were purified and extracted bya solid phase extraction procedure using Oasis?WCX column. The urine was first centrifuged and tak-en out 1 m L into a small column, cleaned by 5% NH4 OH and 20% CH3 CN respectively, eluated usingthe mixture of water and acetomitrile(1/3)with 2% formic acid, blow-dried in the nitrogen at 35℃ andfinally redissolved to be injected into the LC-MS/MS. Result The limits of detection were between0.01~0.5 ng/m L accordingly. The spiked recoveries at the low concentration(1 ng/m L), medium concentration(2 ng/m L)and high concentration(10 ng/m L)ranged between 40% and 76%. The intra-and inter-day precisions of the target substances at these three concentrations were all less than 15%. The indoortemperature, refrigeration condition and multigelation were observed to have significant impact on theanamorelin, GHRP-2,GHRP-4 and GHRP-5. Conclusion The method established in this study is sim-ple, and its specificity and sensitivity meets the international standard and technical documents for labo-ratories set up by the Wworld Anti-Doping Agency. It has been applied in our routine work. Multigela-tion should be avoided in the transport, detection and long-term laboratory storage of urine samples.
引文
[1]https://www.wada-ama.org/en/resources/science-medicine/prohibited-list World Anti-Doping Agency.September29,2015(electronic document).
[2]https://www.wada-ama.org/en/resources/science-medicine/td2014-mrpl World Anti-Doping Agency.Technical Docu-ment for minimum required performance levels[S].Montre-al.September 1,2014(electronic document).
[3]Thomas A,Guddat S,Kohler M,et al.Comprehensive plasma-srenning for known and unknow substances in di-oing controls.Rapid Commun.Mass Spectrom,2010,24(8):1124.
[4]Okano M,Nishitani Y,Sato M,et al.Influce of intrave-nous administration of growth hormone releasing peptide-2(GHRP-2)on detection of growth hormone doping:growth hormone isoform profiles in Japanese male sub-jects.Drug Test Anal,2010,2(11-12):548-556.
[5]Andreas T,Sebastian H,Hans G,et al.Determiantionof growth hormone realeasing peptides(GHRP)and theirmajor metabolites in human urine for doping controls bymeans of liquid chromatography mass spectrometry.AnalBioanal Chem,2011,401:507-516.
[6]Ekaterina Semenistaya,Irina Zvereva,Andreas Thomas,et al.Determination of growth hormone releasing pep-tides metabolites in human urine after nasal administra-tion of GHRP-1,GHRP-2,GHRP-6,Hexarelin,and Ipa-morelin.Drug Test Anal,2015,7(10):919-925.
[7]杨志勇,何根业,陆江海,等.液相色谱-串联质谱法定性检测人尿中八种违禁药物.中国运动医学杂志,2014,33(4):721-724.
[8]申利.高效液相色谱-串联质谱法同时筛查人尿中40种世界反兴奋剂机构禁用药物.体育科学,2015,35(5):66-70.
[9]申利,吴筠,杨志勇,等.兴奋剂检测中吗啡和可待因的LC-MS/MS定量方法研究.中国运动医学杂志,2015,34(11):1089-1093.
[2]https://www.wada-ama.org/en/resources/science-medicine/td2014-mrpl World Anti-Doping Agency.Technical Docu-ment for minimum required performance levels[S].Montre-al.September 1,2014(electronic document).
[3]Thomas A,Guddat S,Kohler M,et al.Comprehensive plasma-srenning for known and unknow substances in di-oing controls.Rapid Commun.Mass Spectrom,2010,24(8):1124.
[4]Okano M,Nishitani Y,Sato M,et al.Influce of intrave-nous administration of growth hormone releasing peptide-2(GHRP-2)on detection of growth hormone doping:growth hormone isoform profiles in Japanese male sub-jects.Drug Test Anal,2010,2(11-12):548-556.
[5]Andreas T,Sebastian H,Hans G,et al.Determiantionof growth hormone realeasing peptides(GHRP)and theirmajor metabolites in human urine for doping controls bymeans of liquid chromatography mass spectrometry.AnalBioanal Chem,2011,401:507-516.
[6]Ekaterina Semenistaya,Irina Zvereva,Andreas Thomas,et al.Determination of growth hormone releasing pep-tides metabolites in human urine after nasal administra-tion of GHRP-1,GHRP-2,GHRP-6,Hexarelin,and Ipa-morelin.Drug Test Anal,2015,7(10):919-925.
[7]杨志勇,何根业,陆江海,等.液相色谱-串联质谱法定性检测人尿中八种违禁药物.中国运动医学杂志,2014,33(4):721-724.
[8]申利.高效液相色谱-串联质谱法同时筛查人尿中40种世界反兴奋剂机构禁用药物.体育科学,2015,35(5):66-70.
[9]申利,吴筠,杨志勇,等.兴奋剂检测中吗啡和可待因的LC-MS/MS定量方法研究.中国运动医学杂志,2015,34(11):1089-1093.