摘要
论文针对鹿茸及其保健品中多种性激素含量的测定，采用液相色谱-串联质谱法（LC-MS/MS）和气相色谱-串联质谱法（GC-MS/MS），研究建立了适用于鹿茸及其保健品中的雄激素、雌激素和孕激素快速测定和确证方法。这些方法填补了国内鹿茸及其保健品中性激素测定方法的空白。
应用LC-MS/MS和GC-MS/MS检测技术，利用多反应监测（MRM）的检测方式，通过对每种性激素的特征目标监测离子的选择，确定了以目标检测物中1对定量离子、2～3对定性离子作为定量和定性检测的依据，优化了质谱条件，考察了方法的分析性能，建立了鹿茸和鹿茸保健品中18种性激素的检测方法。
采用建立的鹿茸及其保健品中18种性激素的快速测定和确证方法，分析了15种鹿茸及其保健品，不仅可了解鹿茸及其保健品中天然性激素的含量情况，而且说明建立的方法有较好的准确度和应用价值。
基于性激素标准品纯度、标准溶液的配制、样品称量、样品萃取、SPE净化、LC-MS/MS检测，对测量不确定度来源进行评估、扩展、合成，计算得到了方法检测结果的扩展不确定度，为检测数据的可靠性提供依据。
Sex hormones can be used to regulate body metabolism and physiologicalfunction and have high biological activity in very low concentrations. The abuse ofsex hormone is harmful to human and animal health, social and ecologicalenvironment. Therefore the detection of sex hormone levels in velvet antler and itshealthy products, especially the simultaneous detection of a variety of sex hormones issignificantly important.
In this thesis18kinds of sex hormones, includingestriol,corticosterone,17β-estradiol,estrone,17α-ethinylestradiol,17α-estradiol,17α-hydroxyprogesterone,medroxyprogesterone,19-nortestosterone,testosteron,megestrol-17-acetate, norethisterone acetate, chloromadinon17-acetate,medroxyprogesterone-17-acetate,progesterone,androsterone,testosteron-17-propionateand17β-Estradiol-benzoate were determined.15kinds of velvet antler and its healthyproducts from home and abroad were analyzed.
The LC-MS/MS was developed to detect sex hormones in velvet antler.Theultrasonic extraction was coupled with solid phase extraction (SPE) to treat to thesamples.The extraction conditions, including extraction solvent type of solid phaseextraction column were examined and optimized. The recoveries are64.17~126.67%and RSDs are lower than20%, the detection limits are0.500~2.00μg/kg. InLC-MS/MS multiple reaction monitoring (MRM) mode was applied.7kinds of velvetantler samples were analyzed, the content of17α-hydroxyprogesterone in the sampleis4.40μg/kg.The content of progesterone in three samples are1.30μg/kg、8.00μg/kgand10.50μg/kg respectively, and the content of testosterone propionate is1.20μg/kgin sample. The analytical results obtained in different laboratories illustrate that themethod has good repeatability.
The LC-MS/MS was developed to detect sex hormones in velvet antler healthyproducts. The ultrasonic extraction was coupled with C18solid phase extractionpurification. The recoveries are71.24~107.40%, RSDs are0.88~16.20%, LODs are0.20~1.50μg/kg.8kinds of velvet antler healthy products were analyzed.17α- hydroxyprogesterone content was10.60μg/kg in one sample and sex hormones inother samples are not detectable.
The GC-MS/MS was developed to detect sex hormones in velvet antler. Theultrasonic extraction was coupled with SPE to treat the samples. The derivativereagent was seven fluorine acetic anhydride and derivative conditions were examinedand optimized. The MRM mode was applied. A precursor/product ion and twoprecursor/product ions were applied for qualitative and quantitative detection,respectively. The recoveries are62.25~95.58%, RSDs are lower than18%, LODs are1.00~30.00μg/kg.7kinds of velvet antler were analyzed, estriol are1.10μg/kg and1.02μg/kg in two samples, respectively.17α-hydroxyprogesterone is4.62μg/kg inone sample. Progesterone are2.51μg/kg、8.64μg/kg and10.40μg/kg in threesamples respectively. LC-MS/MS are not significantly different.
The GC-MS/MS was developed to detect sex hormones in antler velvet healthyproducts. The recoveries are78.17~109.17%, RSDs are1.93~16.47%, LODs are0.50~10.00μg/kg.8kinds of velvet antler healthy products are analyzed.17α-hydroxyprogesterone content was9.90μg/kg in one sample.
When LC-MS/MS was applied the expanded uncertainty is calculated. The effect ofa variety of uncertainties, including sex hormone standard purity, the preparation ofstandard solution, the amount of the sample, sample extraction, purification of SPEand LC-MS/MS determination, on the testing results were examined such as theevaluation, extension and synthesis.Based on sources of measurement uncertainties,the expanded uncertainty was obtained and is0.171.
引文
[1] Sweeley C C, Horning E C.Microanalytical separation of steroids by gas chro-matography[J]. Nature,187(1960)144–145.
[2] Sj vall J, Vihko R.Determination of androsterone and dehydroepiandros-teronesulfates in human serum by gas–liquid chromatography[J].Steroids,6(1965)597–604.
[3] Horning M G, Knox K L, Dalgliesh C E, Horning E C. Gas-liquid chromato-graphic study and estimation of several urinary aromatic acids[J].Anal.Biochem,17(1966)244–257.
[4] Abraham G E.Solid-phaseradioimmunoassayofestradiol[J].Endocrinol.Metab,29(1969)866–870.
[5] Stanczyk F Z, Jurow J, Hsing A W.Limitationsofdirectimmunoassaysfor measuringcirculating estradiol levels inpost menopausal women andmen in epidemiologicstudies[J].CancerEpidemiol.BiomarkersPrev,19(2010)903–906.
[6] Stanczyk F Z, Lee J S, Santen R J.Standardization ofsteroidhormone assays:why,how,andwhen?CancerEpidemiol.BiomarkersPrev,16(2007)1713–1719.
[7] Stanczyk F Z, Cho M M, Endres D B, Morrison J L, Patel S, Paulson RJ.Limita-tions of direc test radiol and testosterone immunoassaykits[J].Steroids,68(2003)1173–1178.
[8] Taieb J, Mathian B, Millot F, Patricot M C, Mathieu E, Queyrel N, Lacroix S I,C,Boudou P.Testosterone measured by10immunoassaysand by isotope-dilutiongaschromatography–mass spectrometry insera from116men,women,andchildren[J].Clin.Chem,49(2003)1381–1395.
[9] Wang C, Catlin D H, Demers L M, Starcevic B, Swerdloff R S.Measurement oftotal serum testosterone in adultmen:comparison of current laboratory methodsversus liquid chromatography–tandem mass spectrometry[J].Clin.Endocrinol.etab,89(2004)534–543.
[10] Sikaris K, McLachlan R I, Kazlauskas R, deKretser D, Holden C A,.Han-delsman DJ.Reproductive hormone reference in tervals for healthy fertile youngmen: evaluation of automated plat for massays[J].Clin.Endocrinol.Metab,90(2005)5928–5936.
[11] Rosner W, Auchus R J, Azziz R, Sluss P M, Raff H.Position statement: util-ity,limitations, and pitfalls in measuring testosterone: an endocrine society positionstatement[J]. Clin. Endocrinol. Metab,92(2007)405–413.
[12] Shackleton C H, Whitney J O. Use of Sep-pak cartridges for urinary steroidextraction: evaluation of the method for use prior to gas chromatographicanalysis[J].Clin. Chem. Acta,107(1980)231–243.
[13] Fenn J B, Mann M, Meng C K, Wong S F, Whitehouse C M. Electrospray ion-ization principles and practice[J]. Mass Spectrom. Rev,9(2005)37–70.
[14] Clarke N J, Rindgen D, Korfmacher W A, Cox K A.Systematic LC/MSmetabolite identification in drug discovery[J].Anal. Chem,73(2001)430A–439A.
[15] Nakamoto J, Fuqua J S. Laboratory assays in pediatric endocrinology: commonaspects[J]. Pediatr. Endocrinol. Rev,5(2007)539–554.
[16] Kushnir M M, Rockwood A L, Bergquist J.Liquid chromatography–tandem massspectrometry applications in endocrinology[J].Mass Spectrom. Rev,29(2010)480–502.
[17] Singh R J.Improving patient care with tandem mass spectrometry[J].Clin. Lab.News,34(2008)12–14.
[18] Wang S, Paris F, Sultan C S, Song R X, Demers L M, Sundaram B.Recombinantcell ultrasensitive bioassay for measurement of estrogens in postmenopausalwomen[J].Clin. Endocrinol. Metab,90(2005)1407–1413.
[19] Xu X, Veenstra T D, Fox S D, Roman J M, Issaq H J, Falk R, Saavedra J E,Keefer L K, Ziegler K G. Measuring fifteen endogenous estrogenssimultaneously in human urine by high-performance liquidchromatography–mass spectrom-etry[J].Anal. Chem,77(2005)7757–7765.
[20] Xu X, Roman J M, Issaq H J, Keefer L K, Veenstra T D, Ziegler R G.Quantitativemeasurement of endogenous estrogens and estrogen metabolites in human serumby liquid chromatography–tandem mass spectrometry[J].Anal. Chem,79(2007)7813–7821.
[21] Vesper H W,Bhasin S, Wang C, Tai S S, Dodge L A, Singh R J, Nelson S,Ohorodnik N J, Clarke N J, Parker C R, Rasdan R, Monsell E A, Myers G L.Interlaboratory comparison study of serum total testosterone measurementsperformed by mass spectrometry methods[J].Steroids,74(2009)498–503.
[22] Annesley T M.Ion suppression in mass spectrometry[J]. Clin. Chem.49(2003)1041–1044.
[23] Rosner W, Vesper H.CDC workshop report improving steroid hormonemeasurements in patient care and translation research[J].Steroids,73(2008)1285.
[24]杨若明，张经华，张林源.麋鹿茸、马鹿茸和梅花鹿茸营养成分的分析比较研究[J].广东微量元素科学，2000，7（12）：47-51.
[25]李和平，丙卜国，郑兴涛，赵景辉，崔贞淑，王忠仁.清原马鹿鹿茸化学成分分析[J].辽宁畜牧兽医，1995，5：7-11..
[26]赵蒙，郑兴涛，李和平，李生，邴国良.东北马鹿乌兰坝品种鹿茸化学成分分析报告[J].经济动物学报，1999，31（1）：28-34.
[27]王丰，梅子青，周秋丽.鹿茸多肽的分离纯化及药理活性[J].吉林大学学报，200301：111-114.
[28]丁玉华，徐安宏，沈华，周元生，徐惠强，孙大明.糜鹿茸化学成分的测定[J].特产研究，1995，1：36-37.
[29]高秀华，李光玉，郜玉钢，王晓伟，王凯英，王俊伟.日粮蛋白质水平对梅花鹿某些相关血液指及鹿茸营养成分的影响[J].特产研究，2000，4：1-7.
[30]肤类兴奋剂检测技术进展[J].体育科技文献通报，2005，13（1）：11-12.
[31]闫谨，王杉，密捷波，常文保.兴奋剂与兴奋剂检测研究进展[J].大学化学，2003，18（2）：11-20.
[32]马生良，王忠武，李海，李生，郑兴涛.兴凯湖农兴凯湖梅花鹿鹿茸几种化学成分分析[J].特产研究，2003，2：39-42.
[1] Ferranti C, Crovella T, Fiori M, Civitareale C。 Simultaneous analysis of17-estradiol and17-estradiol in bovine serum by liquidchromatography–tandem.mass spectrometry[J].Journal of Chromatography B,871(2008)135–140.
[2] Guo T D, Gu J H, Soldin O P., Singh R J., Soldin S J.Rapid measurement ofestrogens and their metabolites in human serum by liquid chromatography-tandemmass spectrometry without derivatization[J].Clinical Biochemistry41(2008)736–741.
[3] Mat D, Kub V.Enhancing sensitivity of liquid chromatographic/ion-trap tandemmass spectrometric determination of estrogens by on-line pre-columnderivatization[J]..Journal of Chromatography A,1192(2008)248–253.
[4] Geisler J, Ekse D, Helle H, Duong N K, Eystein P.An optimised, highly sensitiveradioimmunoassay for the simultaneous measurement of estrone, estradiol andestrone sulfate in theultra-low range in human plasma samples[J].Journal of SteroidBiochemistry&Molecular Biology109(2008)90–95.
[5] Wang S, Huang W, Fang G Z, He J X, Zhang Y.On-line coupling of solid-phaseextraction tohigh-performance liquid chromatography fordetermination ofestrogens in environment analytica[J]. chimica acta,606(2008)194–201.
[6] Koh Y K K, Chiu T Y, Boobis A, Cartmell E, Lester J N, Scrimshaw MD.Determination of steroid estrogens in wastewater by high performance liquidchromatography–tandem mass spectrometry[J].Journal of Chromatography A,1173(2007)81–87.
[7] Yamashitaa K, Okuyama M, Watanabe Y, Honmab S, Kobayashia S, NumazawaM.Highly sensitive determination of estrone and estradiol in human serum by liquidchromatography–electrospray ionization tandem mass spectrometry[J].steroids,72(2007)819–827.
[8] Farr M, Kuster M, Rubio R B F, Alda M J L, Barcel D.Comparative study of anestradiol enzyme-linked immunosorbent assay kit, liquid chromatography–tandemmass spectrometry, and ultra performance liquid chromatography–quadrupole timeof flight mass spectrometry for part-per-trillion analysis of estrogens in watersamples[J].Journal of Chromatography A,1160(2007)166–175.
[9] Labadie P, Hill E M. Analysis of estrogens in river sediments by liquidchromatography–electrospray ionisation mass spectrometry Comparison oftandem mass spectrometry and time-of-flight mass spectrometry[J].Journal ofChromatography A,1141(2007)174–181.
[10] Delvoux B, Husen B, Aldenhoff Y, Koole L, Dunselman G, Thole H, GroothuisP. A sensitive HPLC method for the assessment of metabolic conversion ofestrogens[J]. Journal of Steroid Biochemistry&Molecular Biology104(2007)246–251.
[11] Wang L, Cai Y Q, He B, Yuan C G, Shen D Z, Shao J, Jiang G B. Determinationof estrogens in water by HPLC–UV using cloud point extraction[J].Talanta70(2006)47–51.
[12] Watabe Y, Kubo T, Nishikaw T, Fujita T, Kay K, Hosoy K. Fully automatedliquid chromatography–mass spectrometrydetermination of17_-estradiol in riverwater[J].Journal of Chromatography A,1120(2006)252–259.
[13] Havl L, Nov L, Matysov L, S′cha J, Solich P. Determination of estradiol and itsdegradation products by liquid chromatography[J].Journal of Chromatography A,1119(2006)216–223.
[14] Almeida C, Nogueira J M F. Determination of steroid sex hormones in water andurine matrices by stir bar sorptive extraction and liquid chromatography withdiode array detection[J].Journal of Pharmaceutical and Biomedical Analysis,41(2006)1303–1311.
[15] Xu C L, Chu X G, Peng C F, Jin Z Y, Wang L Y. Development of a fasterdetermination of10anabolic steroids residues inanimal muscle tissues by liquidchromatography tandem mass spectrometry[J].Journal of Pharmaceutical andBiomedical Analysis41(2006)616–621.
[16] Li W K, Li Y H, Li A C, Zhou S L, Weng N D. Simultaneous determination ofnorethindrone and ethinyl estradiol in human plasma by high performance liquidchromatography with tandem mass spectrometry—experiences on developing ahighly selective method using derivatization reagent for enhancingsensitivity[J].Journal of Chromatography B,825(2005)223–232.
[17] Beck C, Bruhn R, Gandrass J, Ruck W. Liquid chromatography–tandem massspectrometry analysis of estrogenic compounds in coastal surface water of theBaltic Sea[J]. Journal of Chromatography A,1090(2005)98–106.
[18] Mitani K, Fujioka M, Kataoka H. Fully automated analysis of estrogens inenvironmental waters by in-tube solid-phase microextraction coupled with liquidchromatography–tandem mass spectrometry[J].Journal of Chromatography A,1081(2005)218–224.
[19] Hu J Y, Zhang H F, Chang H. Improved method for analyzing estrogens in waterby liquid chromatography–electrospray mass spectrometry[J].Journal ofChromatography A,1070(2005)221–224.
[20] Hooijerink H, Lommen A, Mulder P P J, Rhijn J A., Nielen M W F. Liquidchromatography–electrospray ionisation-mass spectrometry based method for thedetermination of estradiol benzoate in hair of cattle[J].Analytica Chimica Acta529(2005)167–172.
[21] Mao L, Sun C J, Zhang H, Li Y X, Wu D S. Determination of environmentalestrogens in human urine by high performance liquid chromatography afterfluorescent derivatization with p-nitrobenzoyl chloride[J].Analytica Chimica Acta,522(2004)241–246.
[22] Mozaz S R, Alda M J L, BarcelóD. Monitoring of estrogens, pesticides andbisphenol A in natural waters and drinking water treatment plants by solid-phaseextraction–liquid chromatography–mass spectrometry[J].Journal ofChromatography A,1045(2004)85–92.
[23] Yoona Y, Westerhoff P, Snyder S A, Esparza M. HPLC-fluorescence detectionand adsorption of bisphenol A,17b-estradiol, and17a-ethynyl estradiol onpowdered activated carbon[J].Water Research37(2003)3530–3537.
[24] Embrechts J, Lemie`re F, Dongen W V, Esmans E L. Detection of EstrogenDNA-Adducts in Human Breast Tumor Tissue and Healthy Tissue by CombinedNano LC-Nano ES Tandem Mass Spectrometry[J].J Am Soc Mass Spectrom2003,14,482–491.
[25] Yamada H, Yoshizawa K, Hayase T. Sensitive determination method of estradiolin plasma using highperformance liquid chromatography with electrochemicaldetection[J].Journal of Chromatography B,775(2002)209–213.
[26] Pen A, Pocurull E, Borrull F, Marce R M. M ethod based on solid-phasemicroextraction–high-performance liquid chromatography with UV andelectrochemical detection to determine estrogenic compounds in watersamples[J].Journal of Chromatography A,964(2002)153–160.
[27] Matsumoto K, Tsukahara Y, Uemura T, Tsunoda K, Kume H, Kawasaki S,Tadano J, Matsuya T. Highly sensitive time-resolved fluorometric determinationof estrogens by high-performance liquid chromatography using ab-diketonateeuropium chelate[J].Journal of Chromatography B,773(2002)135–142.
[28] Alda M, Barcelo D. Use of solid-phase extraction in various of its modalities forsample preparation in the determination of estrogens and progestogens insediment and water[J].Journal of Chromatography A,938(2001)145–153.
[29] Alda M, Barcelo D. Determination of steroid sex hormones and related syntheticcompounds considered as endocrine disrupters in water by fully automated on-linesolid-phase extraction–liquid chromatography–diode array detection[J]. Journal ofChromatography A,911(2001)203–210.
[30] Alda M, Barcelo D. Determination of steroid sex hormones and related syntheticcompounds considered as endocrine disrupters in water by liquidchromatography–diode array detection–mass spectrometry[J].Journal ofChromatography A,892(2000)391–406.
[31] Russell J A, Malcolm R K, Campbell K, Woolfson A D. High-performanceliquid chromatographic determination of17b-estradiol and17b-estradiol-3-acetatesolubilities and diffusion coefficients in silicone elastomeric intravaginal rings [J].Journal of Chromatography B,744(2000)157–163.
[32] Tachibana1S,Tanaka M. Simultaneous Determination of TestosteroneMetabolites in Liver Microsomes Using Column-Switching SemimicrocolumnHigh-Performance Liquid Chromatography[J].Analytical Biochemistry295,248–256(2001).
[33] Houcke S K V，Uytfanghe K V，Shimizu E, Tani W，.IFCC internationalconventional reference procedure for the measurement of free thyroxine inserum[J]. Clin Chem Lab Med2011;49(8):1275–1281.
[34]Schapiro R C，Perrin J，, Roe C M，, Xiong C，Carter D. A Novel UltrapressureLiquid Chromatography Tandem Mass Spectrometry Method for theSimultaneous Determination of Androstenedione, Testosterone, andDihydrotestosterone in Pediatric.Blood Samples: Age-and Sex-Specific ReferenceData[J].Clin Endocrinol Metab95:2399–2409,2010.
[35] Heibeck T H,ing S J, Opresko L K, Zhao R.An Extensive Survey of TyrosinePhosphorylation Revealing New Sites in Human Mammary Epithelial Cells[J].Proteome Res.2009August;8(8):3852–3861.
[36] JIANG J Q, ZHANG L, LI G L，ZHANG H T. Analysis of19-nortestosteroneresidue in animal tissues by ion-trap gas chromatography-tandem massspectrometry[J].Biomed&Biotechnol，2011，12(6):460-467.
[37] Blair I A. Analysis of Estrogens in Serum and Plasma from PostmenopausalWomen: Past Present, and Future [J].Steroids2010April;75(4-5):297–306.
[38] liD W, Stapleton H M. Anlysis of thyroid hormones in serum by liquidchromatography-tandem mass spectrometry[J]. Anal Bioanal Chem2010July;397(5):1831–1839.
[39] Sinclair A J Walker E A, Burdon M l A, Beek A P.Cerebrospinal FluidCorticosteroid Levels and Cortisol Metabolism in Patients with IdiopathicIntracranial Hypertension: A Link between11-HSD1and Intracranial PressureRegulation[J].Clin Endocrinol Metab, December2010,95(12):5348–5356.
[40] Dibble C C, Asara JM,and Manning B D. Characterization of RictorPhosphorylation Sites Reveals Direct Regulation of mTOR Complex2byS6K1[J]. Molecua cell Bio, Nov2009,5657–5670.
[41] Vekiru E, HametnerC Mitterbauer R. Cleavage of Zearalenone by Trichosporonmycotoxinivorans to aNovel Nonestrogenic Metabolite[J].Environ Micro, Apr2010,2353–2359.
[42] Badger J M F, Fuhrman B J, Xu X, Falk R T. Comparison of liquidchromatography-mass spectrometry, radioimmunoassay, and enzyme-linkedimmunosorbent assay methods for measurement of urinary estrogens[J]. CancerEpidemiol Biomarkers，2010January;19(1):292–300.
[43] Sarafoglou K, Himes J H, Lacey J M.Comparison of Multiple SteroidConcentrations in Serum and Dried Blood Spots throughout the Day of Patientswith Congenital Adrenal Hyperplasia[J]. Horm Res Paediatr，2011;75:19–25.
[44] Jonklaas J, Janicic N K, Soldin O P,Correlations of Free Thyroid HormonesMeasured by Tandem Mass Spectrometry and Immunoassay withThyroid-Stimulating Hormone across4Patient Populations[J].ClinicalChemistry，55:71380–1388(2009).
[45] Taylor R L, Grebe S K, and Singh R J Quantitative, Highly Sensitive LiquidChromatography–Tandem Mass Spectrometry Method for Detection of SyntheticCorticosteroids[J]. Clinical Chemistry，50:12,2345–2352(2004).
[46]Earla R, Boddu Si HS, Cholkar K, HariharanS.Development and validation of afast and sensitive bioanalytical method for the quantitative determination ofglucocorticoids-quantitative measurement of dexamethasone in rabbit ocularmatrices by liquid chromatography tandem mass spectrometry[J].Pharm BiomedAnal，2010August1;52(4):525–533.
[47] Zerikly R K, Faiman L A. Gupta M, Singh R J.Diagnostic Characteristics ofLate-Night Salivary Cortisol Using Liquid Chromatography-Tandem MassSpectrometry[J].Clin Endocrinol Metab，95:4555–4559,2010.
[48] Bolca S, Sarda M U, Blondeel P, Roche N. Disposition of soy isoflavones innormal human breast tissue[J].Clin Nutr，2010;91:976–84.
[49] Staniaszek L E, Norris L M, Kendall D A, Barrett D A.Effects of COX-2inhibition on spinal nociception:the role of endocannabinoids[J].British Journalof Pharmacology，2010,160,669–676.
[50] Kenchegowda S, Bazan N G, Bazan E G. Stimulates Lipoxin A4Synthesis andModulates Repair in Corneal Epithelial Cells through ERK and p38Activation[J].IOVS, April2011, Vol52, No52240-2250.
[51] Naessen T, Sjogren U, Bergquist J, Larsson M, Endogenous Steroids Measuredby High-Specificity Liquid Chromatography-Tandem Mass Spectrometry andPrevalent Cardiovascular Disease in70-Year-Old Men and Women[J].ClinEndocrinol Metab,2010，95:1889–1897.
[52] Yue BF, Rockwood A L, Sandrock T, La’ulu S L,Free Thyroid Hormones inSerum byDirect Equilibrium Dialysis and Online Solid-Phase Extraction–LiquidChromatography/Tandem Mass Spectrometry[J].Clinical Chemistry，2008，54:4,642–651.
[53] Rosa F Yeh, Naser L Rezk,Angela D M. Kashuba,Genital Tract, Cord Blood, andAmniotic Fluid Exposures of Seven Antiretroviral Drugs during and afterPregnancy in Human Immunodeficiency Virus Type1-Infected Women.Agentsand chemotherapy, June,2009,2367–2374.
[54] Hughes K A, Reynolds R M, Andrew R, Glucocorticoids Turn Over Slowly inHuman Adipose Tissue in Vivo. Clin Endocrinol Metab95,2010:4696–4702.
[55] Kushnir M M, Rockwood A L, Bergquist Ph D J,High-Sensitivity Tandem MassSpectrometry Assay for Serum Estrone and Estradiol[J].Clin Pathol2008;129:530-539.
[56] Hojo Y, Higo S, Ishii H, Ooishi Y, Mukai H,Comparison betweenHippocampus-Synthesized and Circulation-Derived Sex Steroids in theHippocampus[J].Endocrinology，2009，150:5106–5112.
[57] Kane M A, Folias A E.Attilio Pingitore1Identification of9-cis-retinoic acid as apancreas-specific autacoid that attenuates glucose-stimulated[J].secretion PNAS，50，107，December14,21884–21889.
[58] Bulau P, Meisen, Schmitz T, Keller R Identification of Neuropeptides from theSinus Gland of the Crayfish Orconectes limosus Using Nanoscale On-line LiquidChromatography Tandem Mass Spectrometry[J].Molecular&CellularProteomics，2004，3:558–564,.
[59] Deventer H E, Mendu D R, Remaley A T. Inverse Log-Linear Relationshipbetween Thyroid-Stimulating Hormone and Free Thyroxine Measured by DirectAnalog Immunoassay and Tandem Mass Spectrometry[J].Clinical Chemistry，2011，57:1122–127.
[60] Kushnir M M, Blamires T, Rockwood A L.Liquid Chromatography–TandemMass Spectrometry Assay for Androstenedione, Dehydroepiandrosterone, andTestosterone with Pediatric and Adult Reference Intervals[J].Clinical Chemistry，2010，56:7,1138–1147.
[1]. Matsui F, Kon E, Yamamoto K, Sugimoto K Liquid Chromatography-tandemMass Spectrometry (LC-MS/MS) Assay for Simultaneous Measurement ofSalivary Testosterone and Cortisol in Healthy Men for Utilization in the Diagnosisof Late-onset Hypogonadism in Males[J].Endocrine Journal2009,56(9),1083-1093.
[2]. Shiga N T, Utsunomiya H, Miki Y, Local Biosynthesis of Estrogen in HumanEndometrial Carcinoma through Tumor-Stromal Cell Interactions[J].Clin CancerRes2009;15(19):60,28–34.
[3]. Spink B C, Bennett J A, Pentecost B T, Long-term estrogen exposure promotescarcinogen bioactivation, induces persistent changes in gene expression, andenhances the tumorigenicity of MCF-7human breast cancer cells[J].ToxicolAppl Pharmacol.2009November1;240(3):355–366.
[4] Taylor PJ, Cooper D P, Gordon R D,and StowasserM Measurement of Aldosteronein Human Plasma by Semiautomated HPLC–Tandem Mass Spectrometry.[J].Clinical Chemistry55:6,1155–1162(2009).
[5]. WangC, Catlin D H, Demers L M, Starcevic B, and Swerdloff R S Measurementof Total Serum Testosterone in Adult Men:Comparison of Current LaboratoryMethods Versus Liquid Chromatography-Tandem Mass Spectrometry[J].ClinEndocrinol Metab89:534–543,2004.
[6]. Takagi M, Uno S, Kokushi,E Shiga S, Mukai S,Measurement of urinaryzearalenone concentrations for monitoring natural feed contamination in cattleherds: On-farm trials[J].Anim Sci2011.89:287–296.
[7]. Lin HL, Zhang H, and Hollenberg PF Metabolic Activation of Mifepristone
[RU486;17-Hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-estra-4,9-dien-3-one] by Mammalian Cytochromes P450and the Mechanism-BasedInactivation of Human CYP2B6[J].The American Society for Pharmacology andExperimentalVol.329, No.1329:26–37,2009.
[8]. Lootens L, Meuleman P, Pozo O, J, Eenoou P V.PA/-SCID Mouse withHumanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids:Methandienone as a Case Study[J].Clinical Chemistry55:10,1783–1793(2009).
[9]. Taylor R L, Machacek D, and Singh Rr J. Validation of a High-Throughput LiquidChromatography–Tandem Mass Spectrometry Method for Urinary Cortisol andCortisone[J]. Clinical Chemistry48:9,1511–1519(2002).
[10].Fonseca B M, Correia-da-Silva G, Taylor A H, Lam P M W, N-AcylethanolamineLevels and Expression of Their Metabolizing Enzymes duringPregnancy[J].Endocrinology151:3965–3974,2010.
[11]. Pozo O J, Eenoo V, Deventer K, Lootens L, Thuyne W V. Detection andCharacterization of a New Metabolite of17–Methyltestosterone[J].TheAmerican Society for Pharmacology and Experimental Therapeutics.Vol37, No112153–2162,2009.
[12] Janzen N, Peter,M S Sander, Steuerwald U, Terhardt M, Holtkamp U, andSanderNewborn J Screening for Congenital Adrenal Hyperplasia:AdditionalSteroid Profile using Liquid Chromatography-Tandem MassSpectrometry[J].Clin Endocrinol Metab92:2581–2589,2007.
[13]. Jonkers N, Sousa A,Oliveir S G, Barroso C M Kohler HPE. Occurrence andsources of selected phenolic endocrine disruptors in Ria de Aveiro[J].PortugalEnviron Sci Pollut Res (2010)17:834–843.
[14] Soldin O P, Ph.D, Megan Jang MBA, Guo T, M.Sc,and Soldin S J, PediatricPh.DReference Intervals for Free Thyroxine and Free Triiodothyronine[J].ThyroidVolume19, Number7,2009.
[15]. Ruan CC, Zhu DL, Chen QZi, Chen J.Perivascular Adipose Tissue–DerivedComplement3Is Required for Adventitial Fibroblast Functions and AdventitialRemodeling in Deoxycorticosterone Acetate–Salt HypertensiveRats[J].Arterioscler Thromb Vasc Biol.2010;30:2568-2574.
[16]. Cabaleiro D R, Uytfanghe K V, Stove Ve,Pilot Study for the Standardization ofInsulin Immunoassays with Isotope Dilution–Liquid Chromatography/TandemMass Spectrometry[J].Clinical Chemistry53:8,1462–1469(2007)
[17]. Bystrom C E, Salameh W, Reitz R,and Clarke N J Activity by LC-MS/MS:Development of a Prototypical Clinical Assay Reveals a Subpopulation ofHuman Plasma Samples with Substantial Peptidase Activity[J].ClinicalChemistry56:10,1561–1569(2010).
[18] Hojo S H Y, Ishii H,Endogenous Synthesis of Corticosteroids in theHippocampus[J].Synthesis of Corticosteroids in the HippocampusJuly Issue7Volume62011,21631-21644.
[19] Kumar V, Barnidge D R, Chen LS, Twentyman J M., Cradic K W,Quantificationof Serum1–84Parathyroid Hormone in Patients with Hyperparathyroidism byImmunocapture In Situ Digestion Liquid Chromatography–Tandem MassSpectrometry[J].Clinical Chemistry56:2,306–313(2010).
[20] lkel WV, Bittner N, and Dekant W. Quantitation of bisphenol a glucubonide inbiological samples by high performance liquid chromatography-tandemmassspectrometry[J].The American Society for Pharmacology and ExperimentalTherapeuticsVol33, No11，1748–1757,2005.
[21] Lanza I R, Zhang S, Ward L E, Karakelides H, Raftery D QuantitativeMetabolomics byH-NMR and LC-MS/MS Confirms Altered Metabolic Pathwaysin Diabetes[J].Metabolomics in DiabetesMay Issue5Volume52,10538-10543,2007.
[22] Gjerde J, Geisler J, Lundgren S, Ekse D, Associations between tamoxifen,estrogens, andFSH serum levels during steady state tamoxifen treatment ofpostmenopausal women with breast cancer[J].BMC Cancer2010,10:313.
[23] Kushnir M M, PhD, Ray J A, PhD, Rapid Analysis of25-Hydroxyvitamin D2andD3byLiquid Chromatography–Tandem Mass Spectrometry and Association ofVitamin D and Parathyroid Hormone Concentrations in Healthy Adults[J].Am JClin Pathol2010;134:148-156.
[24] Eliassen A H, Ziegler R G, Rosner B, Veenstra T D Reproducibility of fifteenurinary estrogens and estrogen metabolites over a2-to3-year period inpremenopausal women[J].Cancer Epidemiol Biomarkers Prev.2009November;18(11):2860–2868.
[25]. Aboulfadl K, Potter C D, Time-dependent integrity during storage of naturalsurface water samples for the trace analysis of pharmaceutical products,feminizing hormones and pesticides[J].Chemistry Central Journal2010,4:10.
[26] Roth M Y, LIN K, Amory J K, Matsumoto A M, Anawalt B D Correlates HighlyWith Intratesticular Steroid Levels in Normal Men[J].Androl2010;31(2):138–145.
[27] Shiraishi S, Lee P W N, Leung A, Goh H H, Swerdloff R S,SimultaneousMeasurement of Serum Testosterone and Dihydrotestosterone by LiquidChromatography–Tandem Mass Spectrometry[J].Clinical Chemistry54:11,1855–1863(2008).
[28] Rauh M.,Michael G, and Rascher W Simultaneous Quantification of Ghrelin andDesacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry inPlasma, Serum, and Cell Supernatants[J].Clinical Chemistry53:5,902–910(2007)
[29] Lee S Kwon S, Shin H, Lim H-S, Singh R J, Simultaneous quantitative analysisof salivary cortisol and cortisone in Korean adults using LC-MS/MS[J].BMBreports2010;43(7):506-511.
[30] Fuhrman B J, Pfeiffer R, Xu X, Wu A H. Soy intake is associated with increased2-hydroxylation and decreased16α-hydroxylation of estrogens inAsian-American women Cancer[J].Epidemiol Biomarkers Prev2009October;18(10):2751–2760.
[31] Fuhrman B J, Xu X, Falk RT, Hankinson S E Stability of Fifteen Estrogens andEstrogen Metabolites in Urine Samples under Processing and Storage ConditionsTypically Used in Epidemiologic Studies[J].Int J Biol Markers2010;25(4):185–194.
[32] Thienpont L M, Uytfanghe KV, Blincko S, Ramsay CS, State-of-the-Art ofSerum Testosterone Measurement by Isotope Dilution–LiquidChromatography–Tandem Mass Spectrometry[J].Clinical Chemistry54:8,1290–1297(2008).
[33] Soldin S J Soldin O P,Steroid Hormone Analysis by Tandem MassSpectrometry[J].Clinical Chemistry55:6,1061–1066(2009).
[34] Kushnir M M, Naessen T, Kirilovas D, Chaika A, Nosenko J Steroid Profiles inOvarian Follicular Fluid from Regularly Menstruating Women and Women afterOvarian Stimulation[J]. Clinical Chemistry55:3,519–526(2009).
[35] Kohlen W, Charnikhova T, Liu Q, Bours R, Strigolactones Are Transportedthrough the Xylem and Play a Key Role in Shoot Architectural Response toPhosphate Deficiency in Nonarbuscular Mycorrhizal Host Arabidopsis [J].PlantPhysiology, February2011, Vol155,974–987.
[36] Higashi T, NishioT, Yokoi H, Ninomiya Y, andShimada K.Studies onNeurosteroids XXI: An Improved Liquid Chromatography–Tandem MassSpectrometric Method for Determination of5a-Androstane-3a,17b-diol in RatBrains[J].Analytical sciences august2007, VOL23,1015-1020.
[37] Jonklaasand J, Soldin S J Tandem Mass Spectrometry as a Novel Tool forElucidating Pituitary–Thyroid Relationships[J].Thyroid Volume18, Number12,20081303-1313.
[38] Cawood M L, Field H P, Ford Ce G, GillingwaterS Testosterone Measurement byIsotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry:Validation of a Method for Routine Clinical Practice[J].Clinical Chemistry51:8,1472–1479(2005).
[1] Stopforth A, Burger BV, Crouch A M, Sandra P.The analysis of estrone and17-estradiol by stir bar sorptive extraction–thermal desorption–gaschromatography/mass spectrometry: Application to urine samples after oraladministration of conjugated equine estrogensJournal of Chromatography B,856(2007)156–164.
[2] Arroyo D, Ortiz M C, Sarabia L A.Multiresponse optimization and parallel factoranalysis, useful tools in thedetermination of estrogens by gas chromatography–massspectrometry_Journal of Chromatography A,1157(2007)358–368.
[3] Long Z, Hong L, Jie J.Determination of β-Estradiol Residues in Fish/ShellfishMuscle by Gas Chromatography-Mass Spectrometry. Chin J Anal Chem,2007,35(7),983–987.
[4] Zuo Y, Zhang K, Lin Y. Microwave-accelerated derivatization for thesimultaneous gas chromatographic–mass spectrometric analysis of natural andsynthetic estrogenic steroids. Journal of Chromatography A,1148(2007)211–218.
[5] Choi D W, Kim J Y, Choi S H, Jung H S, Kim H J, Cho S Y, Kang C S, Chang SY. Identification of steroid hormones in omegranate (Punica granatum) usingHPLC and GC–mass spectrometry. Food Chemistry96(2006)562–571.
[6] Kawaguchi M, Ito R, Sakui N, Okanouchi N, Saito K, Nakazawa H. Dualderivatization–stir bar sorptive extraction–thermal desorption–gaschromatography–mass spectrometry for determination of17_-estradiol in watersample. Journal of Chromatography A,1105(2006)140–147.
[7] Basheer C, Jayaraman A, Kee M K, Valiyaveettil S, Lee H K. Polymer-coatedhollow-fiber microextraction of estrogens in water samples with analysis by gaschromatography–mass spectrometry. Journal of Chromatography A,1100(2005)137–143.
[8] Garc′a, Sarabia L A, Ortiz M C, J Aldama M. Optimization of thechromatographic conditions for the determination of hormones by gaschromatography with mass spectrometry detection. Analytica Chimica Acta544(2005)26–35.
[9] Gomes RL, Avcioglu E, Scrimshaw MD, Lester JN. Steroid estrogendetermination in sediment and sewage sludge:a critique of sample preparation andchromatographic/mass spectrometry considerations,incorporating a case study inmethod development. Trends in Analytical Chemistry, Vol.23, No.10–11,2004.
[10] Kawaguchi M, Ishii Y, Sakui N, Okanouchi N, Ito R, Inoue K, Saito K,Nakazawa H.Stir bar sorptive extraction with in situ derivatization and thermaldesorption–gas chromatography–mass spectrometry in the multi-shot mode fordetermination of estrogens in river water samples.Journal of Chromatography A,1049(2004)1–8.
[11] Quintana JB, Carpinteiro J, Rodr′guez I, Lorenzo RA, Carro AM, CelaRDetermination of natural and synthetic estrogens in water by gas chromatographywith mass spectrometric detection. Journal of Chromatography A,1024(2004)177–185.
[12] Fine D D, Breidenbach G P, Price T L, Hutchins S R. Quantitation of estrogensin ground water and swine lagoon samples using solid-phase extraction,pentafluorobenzyl/trimethylsilyl derivatizations and gas chromatography–negativeion chemical ionization tandem mass spectrometry Journal of Chromatography A,1017(2003)167–185.
[13] Lerch O, Zinn P Derivatisation and gas chromatography–chemical ionisationmass spectrometry of selected synthetic and natural endocrine disruptivechemicals. Journal of Chromatography A,991(2003)77–97.
[14] Xiao XY, McCalleya D V, McEvoy J. Analysis of estrogens in river water andeffluents using solid-phase extraction and gas chromatography–negative chemicalionisation mass spectrometry of the pentafluorobenzoyl derivatives. Journal ofChromatography A,923(2001)195–204.
[15] Nakamuraa S, Sian T H, Daishima S. Determination of estrogens in river waterby gas chromatography–negative-ion chemical-ionization mass spectrometry.Journal of Chromatography A,919(2001)275–282.
[16] Edman K, Svensson L, Eriksson B, Gunnarsson PO. Determination ofestramustine phosphate and its metabolites estromustine, estramustine, estrone andestradiol in human plasma by liquid chromatography with fluorescence detectionand gas chromatography with nitrogen-phosphorus and mass spectrometricdetection. Journal of Chromatography B,738(2000)267–279.
[17] Impens S, Loco J V, Degroodt JM, Brabander H D. A downscaled multi-residue
strategy for detection of anabolic steroids in bovine urine using gas
chromatography tandem mass spectrometry (GC–MS3). Analytica Chimica Acta
586(2007)43–48.
[1] Jiang J, Zhang L, Li G., Zhang H, Yang X F. Analysis of19-nortestosteroneresidue in animal tissues by ion-trap gas chromatography-tandem massspectrometry[J].Biomed&Biotechnol，201112(6):460-467.
[2] Courant F, Aksglaede L, Antignac J P, Monteau F, Sorensen K. Assessment ofCirculating Sex Steroid Levels in Prepubertal and Pubertal Boys and Girls by aNovel Ultrasensitive Gas Chromatography-Tandem Mass SpectrometryMethod[J].Clin Endocrinol Metab95:82–92,2010.
[3]. Wang S L, Chang Y C, Chao H R, Li C M, Li L A, Lin L Y, Olaf P. Body Burdensof Polychlorinated Dibenzo-p-dioxins, Dibenzofurans, and Biphenyls and TheirRelations to Estrogen Metabolism in Pregnant Women[J].Environ Perspect May2006Vol114|Num5.740-745.
[4]. Lee J S, Ettinger B, Stanczyk F Z, Vittinghoff E H, Cauley J A. Comparison ofMethods to Measure Low Serum Estradiol Levels in Postmenopausal Women.Environmental Health Perspectives[J]. Clin Endocrinol Metab91:3791–3797,2006
[5]. Verheul H A M, Timmer C J, Van I M L P S,Pharmacokinetic Parameters ofTibolone and Metabolites in Plasma, Urine, Feces, and Bile fromOvariectomized Cynomolgus Monkeys after a Single Dose or Multiple Doses ofTibolone[J].The American Society for Pharmacology and ExperimentalTherapeuticsVol.35, No.71112–1118,2007.
[6]. Low Y L, Taylor J I., Grace P B, Dowsett M, Scollen S. Phytoestrogen ExposureCorrelation with Plasma Estradiol in Postmenopausal Women in EuropeanProspective Investigation of Cancer and Nutrition-Norfolk May InvolveDiet-Gene Interactions[J].Cancer Epidemiol Biomarkers Prev2005;14(1):213–20
[7]. Wang S, Francoise P, Sultan C S, Song R X D, Demers L M, Recombinant CellUltrasensitive Bioassay for Measurement of Estrogens in PostmenopausalWomen[J].Clin Endocrinol Metab90:1407–1413,2005)
[8]. Hsing A W, Stanczyk F Z, Be A, Schroeder P. Reproducibility of Serum SexSteroid Assays in Men by RIA and Mass Spectrometry[J].Cancer EpidemiolBiomarkers Prev2007;16(5):1004–8.
[9]. Yamada M, Aramaki S, Kurosawa A M,Suda I.Simultaneous Doping Analysis ofMain Urinary Metabolites of Anabolic Steroids in Horse by Ion-trap GasChromatography–Tandem Mass Spectrometry[J]. Analytical Sciences September2008, VOL.24111199-1206.
[10]. Swanson C, Mellstro¨m D, Lorentzon M, Vandenput L, Jenny J.The UridineDiphosphate Glucuronosyltransferase2B15D85Y and2B17DeletionPolymorphisms Predict the Glucuronidation Pattern of Androgens and Fat Massin Men[J]. Clin Endocrinol Metab92:4878–4882,2007)
[11]. Ye X Y, Wong L Y, Bishop A M,Calafat A M.Variability of UrinaryConcentrations of Bisphenol A in Spot Samples,First Morning Voids, and24-Hour Collections[J].Environmental Health Perspectives, vol119num7July2011,983-989.
1. Pierre G.Sampling for analytical purposes,translated by A.G.Royle,NewYork:John Wiler and Sons,1998.
2. ISO3082:1998,Iron ores-Sampling and sample preparation procedure,theInternational Organization for Standardization,Geneva,1998.
3. ISO3086:1986,Iron ores-Experimental methods for checking the bias ofsampleing,the International Organization for Standardization,Geneva,1986.
4. EUROCHEM guide,the fitness for purpose of analyticalmethods,EUROCHEM,1998.
5. Currie L A. Limits for qualitative detection and quatitativedetermination:Application to radiochemistry[J].Anal.Chem.,1968,40,586-593.