| The concentration of uE3 can accurately reflect the changes in the fetal placental function. It’s a useful second trimester screening marker in prenatal screening for Down syndrome. And measurement of this analyte is widely used for second-trimester screening for indicate the possibility of premature delivery and fetal preeclampsia. It also commonly used in non-pregnant women screening for cancer epidemiology, breast and cervical cancer risk assessment. There are many routine clinical methods for serum uE3 were developed. Immunoassays are the primary methods for assessing uE3 concentrations in body fluids, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA) and Time resolved f luoroisnmunoassay (TRFIA). However, those methods were having some shortcomings such as poor accuracy and imprecision, low degree of automation and were replaced by chemiluminescence immunoassay (CLIA), gradually. But this widely accepted method has also been shown to lack the specificity and sensitivity necessary to accurately measure low estrogen concentrations. More importantly, since the poor comparability of difference measurement method, which directly affect the credibility of uE3’sMoM in Down syndrome screening.Currently, the reference method of the uE3 measurement has been reviewed by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) is still the GC-MS method.Most of the mass spectrometry methods include uE3 derivatization to volatile components and to increase the sensitivity. Complicated operation procedure requires for high professional skills and is increased the measurement uncertainty. The conventional ID-LC-MS/MS method of the measurement uE3 was been developed by my tutor, and the method was used to evaluated two different CLIAsystems. It was been found that there are obvious systematic errors between the two systems. Therefore, the establishment of uE3 reference method is very important. In addition, there are no reference materials of uE3 in our country and abroad, and the reference interval was established by Siemens and only a small sample of 15 foreign people, no Chinese reference intervals.In this background, to establish a candidate reference method by use of ID-LC-MS/MS technique that has high sensitivity and specificity.This research conducted a comprehensive study for the pre-treatment, chromatographic and MS conditions, and a comprehensive evaluation of the method performance on the basis of established uE3 conventional ID-LC-MS/MS method. By use of candidate reference method to establish secondary reference materials and serum reference interval study of Chinese population. To assess the results of the reference measurement uncertainty which built a complete uE3 reference system. After the reference system was been established, it can be used to evaluate the conventional CLIA systems. This study can provide a technology platform for uE3 measurement standardization and also provide a reference for other hormones.ObjectiveTo optimize the pretreat conditions of the determination of unconjugated estriol (uE3) in human serum using isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC-MS/MS). To evaluate the analysis performance of the candidate reference method and verfify whether it meet the requirement of reference method. To establish a comprehensive reference system and standardization process though the preliminary application by using the candidate reference method.MethodsEstriol (purity>99.3%) and estriol-2-3-4-13C3 were the standard and the internal standard, respectively. Activated carbon adsorption serum was used for the study of serum matrix. We optimized the liquid chromatography (LC)and mass spectrometry (MS) condition and pretreat condition including extraction, iron suppression, equilibration and matrix effect to increase the sensitivity of the detection by use of the the AB/Sciex API 5500 tandem mass spectrometer equipped with Shimadzu LC-20AD XR system.We used calibrator bracketing to give higher specificity and accuracy to serum value assignments. The accuracy of the candidate RMP was evaluated by use of a recovery study for the added uE3 and validated by split-sample comparison to established RMPs. The intraassay and interassay imprecision, sensitivity, specificity, potential interferences, matrix effects, ion suppression, carryover and sample stability were evaluated.The serum samples from patients in the second trimester of pregnancy were used to measure uE3 concentrations by this LC-MS/MS method and Siemens IMMULITE 1000 systems, Auto DELFIA1235, and Beckman Coulter Access 2. A candidate reference material was prepared and evaluated for its homogeneity, commutability and stability. Furthermore,the reference interval of male and nonpregnant and pregnant female would be established.ResultsMS condition:the samples were analyzed by ID-LC-MS/MS system in the negativelyelectro-spray ionization (ESI) mode and multiple reaction monitor(MRM)mode. And the MRM transitions used for each analytes were:(m/z) 287â†'145 (quantitative) and m/z 287â†'171 (qualitative) for uE3, m/z 290â†'148 (quantitative) and m/z 290â†'174 (qualitative) for internal standard.LC condition:Knietex 100×2.1 mm,2.6μm as the column, the mobile phase was fluoride (NH4F) in water (buffer A) and mcthanol (buffer B). The analysis of uE3 was accomplished within 8 min by T7 model.Pretreat condition:n-Hexane/ethyl acetate (50:50, v/v) was chosen to the extraction solvent, and the volume ratio of the serum sample to extraction solvent was 1:2. The mean extraction efficiency for the method was determined to be 85.71%. Evaluation of the suppression not showed reduction (less than 5%) in the specific signal for uE3.30 min was chosen as the time for equilibration.Method performance:The candidate RMP gave intraassay and interassay CVs of 0.45%-1.36% and 0.66%-2.04% and across the concentrations of uE3 at 0.4-20. Ong/mL. Recovery of the added uE3 was 99.2%. The results of PT and RELA were agreed with the target values. LOD was 0.04 ng/mLand LOQ was 0.1ng/mL. Linear range was 0.lng/mL-50ng/mL. Two of the eleven structural analogs 16-epiestriol (molecular mass 288.39) and testosterone (molecular mass 288.42) having the same molecular mass with uE3 which could be detected in the MS conditions use for uE3 measurement. But they were completely separated from uE3 under the LC conditions. The other nine structural analogs were none peak at the retention time for uE3 there are no endogenous compounds interferences and provides a true value in this candidate RMP. Carryover was less than<0.1%.Application of method:Method comparison showed up to 37% positive bias that gradually decreased as the concentration increased.The candidate reference material has good homogeneity and commutability and it was meet the requirements of the stability study, which variaton was less than 2% under the freezing condition.The measurement uncertainty was 2.48%, which was better than other certificated reference materials for other harmone. The referece interval of male and nonpregnant female was below 0.1 ng/mL.ConclusionsThis study successfully optimized the LC and MS conditions and the ideal condition was obtained of the determination of uE3 in human serum using ID-LC-MS/MS. The applicable extract method was determined. It was an important base of establish the candidate reference measurement procedure for the determination of uE3 in human serum using ID-LC-MS/MS.This method provides highly accurate and precise value, and ensures accurate and comparable results across testing systems and laboratories. This candidate reference method was found to be free from interferences by testing lipemic, hemolytic, and icteric samples and by testing the structure analogues under the same assay conditions for uE3 measurement.A set of standard determination procedure for the uE3 has been established, which including reference material, reference interval, MU determination based on candidate reference method, and which can also provide a reference for standardization of other tests. |
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