人血浆中类固醇检测方案(液相色谱仪)

2 4 Baptiste Grund， Hugues Henry?， Alexandre Beguin?， Maciej Bromirski， Bertrand Rochat； 1Quantitative MassSpectrometry Facility， CHUV University Hospital of Lausanne， Lausanne， Switzerland. 2Biomedicine， CHUVUniversity Hospital of Lausanne， Lausanne， Switzerland. 3Thermo Fisher Scientific， Bremen， Germany Steroids，LC-HRMS， plasma， Q Exactive Focus， quantification To develop a robust liquid chromatography-high-resolution massspectrometry (LC-HRMS) analysis for the routine quantification of steroids inplasma that fulfills clinical research demand for sensitivity and selectivity. Introduction Steroid hormones are endogenous metabolites thatare synthesized by various enzymes from cholesterol.There are tens of these hormones with different effectseven at very low concentrations (nM-pM levels).1.2Their determination can be relevant for clinical research.Steroid determinations by immunoassays are affected byinterferences and are not fully appropriate for childrenand women. The steroid backbone of cholesterol does not containamino groups； therefore， various ionization sources havebeen employed.However， recent routine LC-MS researchanalyses of steroids (underivatized) use a 2.1 mm ID(inner diameter) analytical column coupled withelectrospray ionization (ESI) and triple quadruople MS. Indeed， LC-MS research methods almost exclusively usetriple-quadrupole mass spectrometry. However， manyarticles have recently shown that LC-MS analysis usinghigh resolution (HR) technology can perform robust，quantitative and sensitive analyses of drugs and peptidesin routine research environments. Interestingly， mostLC-HRMS analyses are performed in HR full scanacquisition which allows an overview of all ions such assteroid metabolites. The work presented here is to analyze quantitativelyeight steroids using an affordable HRMS instrument， theThermo ScientificTM Q ExactiveTM Focus MS， for clinicalresearch purposes. Experimental Chemicals Cortisol， corticosterone， 11-deoxycortisol，deoxycorticosterone， androstenedione， 17-hydroxyproges-terone， and progesterone standards were purchased fromSigma-Aldrich chemie GmbH. Internal standards (IS)，d，-progesterone， d-cortisol， d -corticosterone，d，-11-deoxycortisol， d -deoxycorticosterone， andd -17-hydroxyprogesterone were purchased from C/D/NIsotopes Inc. D-androstenedione and d，-testosterone werepurchased from Cambridge Isotope Laboratories， Inc. andLipomed AG， respectively. Sample Types Human plasma samples were prepared by centrifugationof donor whole blood withdrawn in heparin tubes. Plasma samples were stored at -80 °C. Two qualitycontrol samples (QCs)， one as a pool of female plasmaand one as a pool of male plasma samples， were prepared.Calibrators (Cs) were prepared from stripped fetal bovineserum and fortified with seven different concentrations(see Table 1). Sample Preparation Plasma samples (100 pL) were diluted with 100 pL of5% phosphoric acid (H，PO) containing the internalstandards (IS) in water. The solution was stirred for10 minutes. A solid phase extraction (SPE) was performedusing 96-well plates (mixed mode cation extraction96-well plate， 10 mg of phase). The SPE plate wasactivated with 200 uL of methanol (MeOH) and 200 uLof water. The samples were loaded on the plate withpositive pressure， washed with 200 pL of 5% ammoniumhydroxide (NHOH) and 200 pL of 10% MeOH in water.Then， the steroids were eluted with 2 x75 pL ofisopropanol. The wells were dried under nitrogen flowand the dried residues were reconstituted in 100 pL ofH，O/MeOH (50：50) prior injection. LC Conditions The LC system consisted of a Rheos Allegro UHPLCpump (Flux Instruments， Basel， Switzerland) and aCTC PAL autosampler (CTC Analytics， Switzerland).A Thermo ScientificTM AccucoreTM C18 LC column(2.1 x50 mm and 2.6um， P/N 17126-052130) was usedand placed in an oven set at 60C. Mobile phase wascomposed of A) H，O and B) MeOH with 50 pL/L offormic acid. The gradient was delivered as follows： at0 min： 10% of B； at 15 min： 75% B；at 15.10 min： 100%of B kept to 17.90 min； 18 min： 10%of B and maintainisocratic to 20 min for column initial conditions. The flowrate was 600 pL/min. The injection volume was 40 pL. MS Conditions The LC system was connected to a Q Exactive Focushigh-resolution mass spectrometer； the followingMS conditions were used： Source type Heated-electrospray ionization (HESI II) lonization mode Positive Spray voltage 4000V Sheath gas， N2 60AU Sweep gas， N2 20 AU S lens 70V Capillary temperature 380 °℃ Auxiliary gas heater Deactivated Data acquisition mode HR full scan Scan range m/z 200 to 400 Resolution 70，000 (FWHM) at m/z 200 A second acquisition was studied and consisted of anHR full scan (same range and resolution) and a targetedsingle ion monitoring (SIM) scan， which allowed thedetection of both testosterone and d.-testosterone in thesame SIM scan： SIM scan Centered on m/z 290 |solation window 8m/z C-trap capacity 5x 105 charges Maximum injection time 100 ms Results and Discussion The determined steroids are presented in Table 1 andwere detected as singly charged molecular ions [M+H]t.Extracted ion chromatograms (XIC) were constructed onthe theoretical m/z (m/Ztheor； Table 1) with a massextraction window of±5 ppm. Table 1. Steroids determined， chemical composition， m/z used for XIC construct， retentiontime， and the lowerand upper calibrator levels (Cs). Name Formula m/Z theor[M+H]+ R- [min] Lower CsnM] Upper CsnM] Cortisol CH0， 363.21660 8.5 2.19 1400 Corticosterone C，H0. 347.22169 9.8 0.47 60 11-Deoxycortisol CHO 347.22169 10.1 0.16 20 Deoxycorticosterone CH0. 331.22677 11.8 0.16 20 Androstenedione C262 287.20056 10.7 0.31 40 Testosterone CH280 289.21621 11.3 0.13 80 17-hydroxyprogesterone CH0 331.22677 11.2 0.16 20 Progesterone C，H0， 315.23186 13.3 0.50 160 Typical XIC traces constructed from HR full scanacquisition are presented in Figure 1 and show theresolution of many steroid isomers in donor plasma in lessthan 14 minutes. Similar chromatograms presentingvarious isomers have been shown with SRMacquisitions46revealing that an efficient chromatographicresolution， typically using UHPLC or core shell columns，is mandatory. Figure 1. Example of XIC chromatograms extracted from HR full scan acquisition. Left： a male donor plasma sample. Right： a calibratorsample (stripped plasma fortified). Extracted m/z is listed on the right. Calibration curves and equations were generated usingthe internal standard methodology. An example of acalibration curve is depicted in Figure 2. Analyses werevalidated according to international guidelines. Accuracyof calibrators， QCs， and lower limit of quantification(LLOQ) levels were <15% and <20%， respectively. To determine the LLOQ for testosterone， HR full-scanand tSIM mode data were acquired successively. VariousCs at expected LLOQ values (N =5 extracts) included ina full calibration (with the upper limit of calibrations=80 nM) were injected. Generic ESI source parameterswere employed (no specific adjustments of the ESI sourceparameters were made for testosterone or anysteroids tested). Figure 2. Left： Calibration curves of testosterone in fortified stripped plasma. Right： Corresponding XIC at the LLOQ level in plasma fortestosterone (0.125 nM). Top and bottom： Data according to HR full-scan ortargeted SIM acquisitions， respectively. In HR full-scan acquisition on the Q Exactive Focus MSsystem， the LLOQ value for testosterone in plasmasamples (volume=100 pL) was 0.125 nM with aprecision and accuracy of 19% and 8%(N=4)，respectively； whereas， in SIM mode，precision andaccuracy were 21% and 7%， respectively. SIM acquisitiondid not significantly increase the LLOQ level (still at0.125 nM). However， the limit of detection (LOD) fortestosterone was increased by about five times in the SIMmode (not shown). As an illustrative comparison only， the LLOQlevels (nM)and amounts (pg) on column at the LLOQ levels ofsteriods by ESI-MS in eleven published articles arepresented in Table 2. Mean ±SD of the amounts oncolumn at the LLOQ values is 1.77± 1.22 pg. Generic ESIparameters were used. Our LLOQ levels could probablybe improved by adjusting ESI parameters for testosterone(e.g. heated gas temperature， etc.)or by extracting abigger plasma volume (e.g. 200-400 pL instead of100 pL). Nevertheless， even without tuning of parametersfor sensitivity， the results show that the Q Exactive FocusMS has a comparable sensitivity to triple quadrupoleinstruments (Table 2). The amount on column at theLLOQ value was 1.45 pg. The obtained sensitivityallowed performing steroid determination in all donorsamples. Conclusion A robust and rapid LC-MS method for the determinationof steroids in plasma samples using an affordable HRMSinstrument has been presented and is suitable for routineresearch analyses. Results acquired using this methoddemonstrate that the Q Exactive Focus MS exhibitsaccuracy， precision， and sensitivity that is comparable totriple quadrupole MS instruments. Various steroid isomerscan be detected in full scan but also in MS/MS acquisitionshowing that there is a need for an efficientchromatography. In addition， with sensitive and selectivefull scan data， the Q Exactive Focus HRMS can be usedfor more in-depth investigations when requested byresearchers or biochemists. Table 2. LLOQ levels in plasma/serum matrices and amounts (pg) on column for testosterone in routineanalyses in research found in the literature. HR-MS full scan (#7) and SRM (#1 to #6 and #8 to #13)acquisitions on various MS platforms can be compared but are illustrative only. The comparison shouldbe taken with care because different LLOQ determinations， extraction procedures and yield， columns， ionsources，LC-MS conditions，S/N ratios， etc. have been used. ReferenceOrdered by pg on column Instrument pg oncolumn (*) LLOQ [nM](**) 1. Salameh et al.， 20107 TSQ Ultra， Thermo Scientific 0.45 0.01 2.Han et al.，20148 QTRAP 5500， AB Sciex 0.50 0.13 3.Rhea et al.， 2013° QTRAP 5000， AB Sciex 0.60 0.03 4.Wang et al.，2014 API5500， AB Sciex 0.75 0.03 5.Speborg et al.， 2013 TSQ Vantage， Thermo Scientific 1.10 0.10 6.Buttler et al.， 2015" Xevo TQS， Waters 1.16 0.10 7.Presented data， 2015 Q Exactive Focus MS， Thermo Scientific 1.45 0.13 8.Rhea et al.. 20139 QTRAP 4000， AB Sciex 1.60 0.06 9.Koal et al.，20122 QTRAP 4000， AB Sciex 2.00 0.03 10. Keski-Rahkonen et al.， 201113 Agilent 6410， Agilent 2.60 0.08 11. Kyriakopoulou et al.， 201314 QTRAP 4000，AB Sciex 3.00 0.08 12. Ke et al.， 20144 QTRAP 6500， AB Sciex 3.75 0.17 13. Koren et al.， 20125 QTRAP 5500， AB Sciex 4.00 0.35 (*)： according to a 100% extraction yield ( References ) ( 1. Ketha，H . ； K a ur， S.； Grebe， S.K.； Singh， R.J. Clinical applications of LC-MS sex steroid assays： evolution of methodologies in the 2 1st century. Curr Opin Endocrinol Diabetes Obes. 2 014，21，217-26. ) ( 2. Greaves， R.E； Jevalikar， G.； Hewitt， J.K.； Zacharin， M.R. A guide to understanding th e steroid pathway： new insights and diagnostic implications.Clin Biochem.2014，47，5-15. ) ( 3. Rochat， B.； Kottelat， E.； McMullen， J. The future keyrole of LC-high-resolution-MS analyses in clinicallaboratories： a focus on quantification. Bioanalysis.2012，4，2939-58. ) ( 4 . K e，Y.； B ertin，J； G onthier， R.； Simard， J.N.； Labrie， F. A sensitiv e ， simple and robust LC-MS/MS method for the simultaneous quantification of seven androgen- and e strogen-related steroids i n postmenopausal serum. J Steroid Biochem Mol Biol. 2014， 144，523-34. ) ( 5. Koren， L.； Ng， E.S.； Soma， K.K.； Wynne-Edwards， K.E. Sample preparation a nd liquid chromatography- tandem mass spectrometry for multiple steroids inmammalian and avian circulation. PLoS One.2012，7：e3249. ) ( 6. S oeborg，T.； Frederiksen， H.； Fruekilde， P.；Johannsen，T.H.； Juul， A.； Andersson， A.M. Serum c oncentrationsof DHEA， DHEAS， 17o-hydroxyprogesterone， A4-androstenedione and t e stosterone in childrendetermined by TurboFlow-LC-MS/MS. Clin Chim Acta.2013，419，95-101. ) ( 7. Salameh， W.A.； Redor-Goldman， M.M.； Clarke， N.J； Reitz，R.E.； Caulfield， M.P. Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry： total and f ree testosterone reference ranges.Steroids. 2010，75，169-75. ) ( 8. Han，J； Zhang， S.； Liu， W.； Leng， G.； Sun， K.； Li， Y.； Di， X. An analytical s trategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC-MS/MS analysis of triptorelin and endogenous te s tosterone in ra t plasma. Anal Bioanal Chem. 2014，406，2457-65. ) ( 9. Rhea，J.M.； French， D .；Molinaro， R.J. Direct total and free testosterone measurement by liquid chromatography tandem m a ss s p ectrometry across two d ifferent platforms. Clin Biochem. 2013， 46，656-64. ) 10. Wang， Y.； Gay， G.D.； Botelho， J.C.； Caudill， S.P.；Vesper， H.W. Total testosterone quantitativemeasurement in serum by LC-MS/MS.Clin Chim Acta.2014，436，263-7. 11.Buttler， R.M.； Martens，F； Kushnir， M.M.；Ackermans， M.T.； Blankenstein， M.A.； Heijboer， A.C.Simultaneous measurement of testosterone，androstenedione and dehydroepiandrosterone(DHEA) in serum and plasma using Isotope-Dilution2-Dimension Ultra High Performance Liquid-Chromatography Tandem Mass Spectrometry(ID-LC-MS/MS).Clin Chim Acta.2015，438，157-9. 12. Koal， T.； Schmiederer， D.； Pham-Tuan， H.； Rohring，C.； Rauh， M. Standardized LC-MS/MS based steroidhormone profile-analysis.J Steroid Biochem Mol Biol. 2012，129，129-38. ( 13.Keski-Rahkonen， P.； Huhtinen， K . ； Poutanen， M. ； Auriola， S. Fast and sensitive liqui d chromatography-mass s pectrometry assay for seven androgenic andprogestagenic steroids in human serum. J Steroid Biochem Mol Biol. 2011， 127， 396-404. ) ( 14. Kyriakopoulou， L.； Yazdanpanah， M.； Colantonio，D.A.；Chan， M.K.； Daly， C.H.； Adeli， K. A sensitiveand rapid mass spectrometric method for thesimultaneous measurement of eight steroid hormones and C ALIPER pediatric reference in t ervals. Clin Biochem. 2013，46，642-51. ) ( For research use only. Not for use in diagnostic procedures. ) www.thermofisher.com ◎2016 Thermo Fisher Scientific Inc. All rights reserved. PAL is a registered trademark of CTC Analytics AG. All other trademarks are theproperty of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo FisherScientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights ofothers. Specifications， terms and pricing are subject to change. Not all products are available in all countries. Please consult your local salesrepresentative for details. A Thermo Fisher Scientific Brand S CIENTIFIC ANEN To develop a robust liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis for the routine quantification of steroids in plasma that fulfills clinical research demand for sensitivity and selectivity.Steroid hormones are endogenous metabolites that  are synthesized by various enzymes from cholesterol. There are tens of these hormones with different effects even at very low concentrations (nM–pM levels).1,2   Their determination can be relevant for clinical research. Steroid determinations by immunoassays are affected by interferences and are not fully appropriate for children and women.The steroid backbone of cholesterol does not contain amino groups; therefore, various ionization sources have been employed. However, recent routine LC-MS research analyses of steroids (underivatized) use a 2.1 mm ID (inner diameter) analytical column coupled with  electrospray ionization (ESI) and triple quadruople MS.Indeed, LC-MS research methods almost exclusively use triple-quadrupole mass spectrometry. However, many articles have recently shown that LC-MS analysis using high resolution (HR) technology can perform robust, quantitative and sensitive analyses of drugs and peptides in routine research environments.3 Interestingly, most LC-HRMS analyses are performed in HR full scan acquisition which allows an overview of all ions such as steroid metabolites. The work presented here is to analyze quantitatively  eight steroids using an affordable HRMS instrument, the Thermo Scientific™ Q Exactive™ Focus MS, for clinical research purposes.A robust and rapid LC-MS method for the determination of steroids in plasma samples using an affordable HRMS instrument has been presented and is suitable for routine research analyses. Results acquired using this method demonstrate that the Q Exactive Focus MS exhibits accuracy, precision, and sensitivity that is comparable to triple quadrupole MS instruments. Various steroid isomers can be detected in full scan but also in MS/MS acquisition showing that there is a need for an efficient chromatography. In addition, with sensitive and selective full scan data, the Q Exactive Focus HRMS can be used for more in-depth investigations when requested by researchers or biochemists.