| Steroid hormone is a group of lipid-soluble hormones that has the basic cyclopentanoper-hydrophenanthrene ring structure.The measurements of steroid hormone are of central importance to the practice of clinical endocrinology.Cortisol,also known as hydrocortisone,is a primary indicator of adrenal cortex function.Testosterone is the primary male sex hormone.The routine methods for serum steroid hormones are mainly based on immunoassay,including chemiluminescent immunoassay,radioimmunoassay,electrochemiluminescence assays,et al,as well as clinical mass spectrometry which has been developing rapidly in recent years.However,according to the statistics from national and other country or regions' external quality assessment(EQA)schemes,the performance of routine serum cortisol and testosterone testing remain highly variable,especially in pregnant female,patient taking exogenous glucocorticoids or adrenal steroidogenesis inhibitors.Reliable measurements of serum cortisol and testosterone that are comparable over time and location and across assays are requisite for proper diagnosis and treatments,therefore it is necessary to realize the standardization of routine cortisol and testosterone assays.For this to be realized,it is essential to have a higher order method to provide an accuracy base to which routine methods can be compared.The majority of cortisol and testosterone circulates bound to protein in serum,with only a small fraction of circulating in the free(unbound),biologically active form.Because of their low level,the complexity of serum matrix and interfering analogs,it will be very challenging to establish the highly reliable reference methods for serum cortisol and testosterone.According to Joint Committee on Traceability in Laboratory Medicine(JCTLM)list,only a few institutions had established the reference measurement procedures(RMPs)for serum cortisol and testosterone.The existing RMPs are based on isotope dilution gas chromatography-mass spectrometry(ID GC-MS)or isotope dilution liquid chromatography-tandem mass spectrometry mass spectrometry(ID LC-MS/MS),with liquid-liquid extraction,gel-filtration chromatography,solid phase extraction,derivatization,2D-chromatography or their combinations as sample pretreatment procedures.The existing RMPs have been successfully applied in the standardization of serum cortisol and testosterone measurements.These procedures were required multistep operations before achieving a concentrated extract suitable for instrumental analysis,which could be costly,cumbersome and time-consuming.The RMPs for serum cortisol and testosterone has not yet been fully established in our country.In the first section of this study "the establishment of a candidate reference measurement procedure for serum cortisol using isotope dilution chromatography tandem mass spectrometry",a new sample pretreatment method based on lipoproteins precipitation with dextran sulfate-Mg2+,which was inspired by our previous work on measurements of high-density lipoprotein cholesterol,was investigated.The sample was pre-purified by lipoprotein precipitation before conventional solvent-solvent extractions.The within-run and total CVs ranged from 0.3%to 0.6%,and 0.7%to 1.2%,respectively,for a concentration range of 76.30 to 768.04 nmol/L.A regression comparison of the results obtained by present method and the certified values of ERM-DA451 showed agreement withno statistical difference(Y=1.0092X-0.7455;95%CI for the slope,0.9940 to 1.0212;95%CI for the intercept,-3.6575 to 2.6390,r2=0.999).All structural analogs of cortisol tested were well resolved from cortisol in 12 min on a phenyl ligand column under an isocratic elution.The limit of quantification was estimated to 5 pg cortisol in absolute amount.This method used for participation in the IFCC ring trial for reference laboratories(RELA)for cortisol.The data were in good agreement with results of other participating laboratories.This method is accurate,simple and can be served as a candidate reference measurement procedure in establishment of serum cortisol reference system.In the second section "the establishment of a candidate reference measurement procedure for serum testosterone using isotope dilution chromatography tandem mass spectrometry",a simple procedure for the extraction and purification of testosterone was established,which involved an extraction of serum with organic solution and 2-hydroxypropyl-?-cyclodextrin(HP-?-CD).The investigations on equilibration,preparation of calibrators,optimization of LC-MS/MS analysis were conducted to develop a candidate reference method for the testosterone measurements.In the present method,the within-run,between-run and total coefficients of variation ranged from 0.22%to2.00%,0.48%to 2.37%and 0.53%to 3.09%,and the averages were 0.84%,1.01%and 1.37%,respectively.The results of analyzing the certified reference material IRMM ERM DA-345a and NIST SRM 971 showed biases ranged from-2.0%?+0.8%.The limit of quantification was estimated to 8 pg testosterone in absolute amount.No apparent interference was observed.This method was used for participation in the IFCC RELA for testosterone.The data were in good agreement with results of other participating laboratories.This method is accurate,simple and can be served as a candidate reference measurement procedure in establishment of serum testosterone reference system.In the third section "the application of the RMPs for serum cortisol and testosterone",the first national trueness verification schemes for steroid hormones were running with the candidate RMPs as the methods for target value assignment.The survey sample were prepared from patient sera which showed good commutability with native samples.It demonstrated that the samples were homogenous and stable under-70? for at least 28 months.The survey samples were shipped by dry ice to participating laboratories including 50 clinical laboratory using immunoassays and 50 ones using mass spectrometry.The samples were measured repeatedly as required.The results showed that the average deviation of the trimmed means of two survey samples from the target values assigned by the developed methods for serum cortisol were-12.4%?+17.8%,the variation coefficients(CVs) for different analytical instrument platform were 3.7%?21.3%.The average deviation of the trimmed means of two survey samples from the target values for serum testosterone were-33.4%?+5.5%,the CVs were 3.8%?11.4%.The performance of serum cortisol and testosterone at clinical laboratory still has a room for improvement.In summary,we developed and validated the highly reliable ID LC-MS/MS measurement procedures for serum cortisol and testosterone with the new sample pretreatment process based on lipoproteins precipitation and purification with HP-?-CD,respectively.They all showed good accuracy and precision performance compared to existing RMPs.The first national trueness verification schemes for steroid hormones were conducted and the state-of-the-art performance of serum cortisol and testosterone at clinical laboratory in China was assessed The present candidate reference measurement procedures are simple,precise,and accurate,can be served as the essential tools in establishment of serum cortisol and testosterone standardization in China. |
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