| Objective: To validate ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method for measuring serum Testosterone(T),Dehydroepiandrosterone Sulfate(DHEA-S),Androstenedione(AD)and 17?-hydroxy progesterone(17?-OHP)levels in human blood samples.To compare UPLC-MS/MS and Chemiluminescence Immunoassay(CLIA)measurement of human serum androgen levels in Polycystic Ovarian Syndrome(PCOS).Method: 160 PCOS patients and 146 healthy subjects(control group)were recruited from Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine.PCOS was diagnosed according to the Revised Criteria of Rotterdam(2003).Referring to Clinical and Laboratory Standards Institute(CLSI)evaluation protocols,the lower limit of detection,precision,analytical measurement range,reference interval and recovery of T,DHEA-S,AD and 17?-OHP tested by UPLC-MS/MS were validated.100 of these specimen were also picked up to test for T,DHEA-S,AD and17?-OHP by UPLC-MS/MS,and the results were compared in the same specimen using either CLIA or Radioimmunoassay(RIA).After method validation,the patients and controls' serum androgen levels--testosterone(T)and dehydroepiandrosterone sulfate(DHEA-S)were tested by UPLC-MS/MS and CLIA.The androgen results combined with body mass index(BMI)were analyzed by Receiver Operating Characteristic Curve(ROC Curve).According to the area under the curve(AUC)calculated by SPSS,a better method will be selected to provide accurate test results for physicians to diagnose PCOS.Results: Method validation results:(1)Lower limit of detection of T,DHEA-S,AD and17?-OHP was 0.02ng/ml,5ng/ml,0.02ng/ml and 0.02ng/ml,respectively.(2)Within-run and total coefficients of variation(CV)of T were 4.86%-5.6% and5.53%-6.17%,within-run and total CV of DHEA-S were 2.58%-5.31% and2.88%-7.18%,within-run and total CV of AD were 4.81%-5.2% and 4.55%-5.89%,within-run and total CV of 17?-OHP were 3.66%-4.79% and 3.58%-4.98%.(3)Analytical measurement range of T,DHEA-S,AD and 17?-OHP was 0.02-100ng/ml,5-4000ng/ml,0.02-100ng/ml and 0.02-100ng/ml,respectively.(4)Reference interval of T,DHEA-S,AD and 17?-OHP was 0.15-0.63 ng/ml,423-1929 ng/ml,0.94-4.25ng/ml and 0.03-2.73 ng/ml,respectively.(5)The recovery of T,DHEA-S,AD and17?-OHP was within 80-120%.(6)The test results showed UPLC-MS/MS was significantly positively associated with CLIA and UPLC-MS/MS was positively associated with RIA.Comparison of UPLC-MS/MS and CLIA measurement of human serum androgen levels in PCOS:(1)The AUC(0.789)of DHEA-S tested by UPLC-MS/MS was significantly higher than the one(0.661)tested by CLIA(P<0.01).There was no significant difference between the AUC(0.85)of T tested by UPLC-MS/MS and the one(0.805)tested by CLIA.(2)The AUC(0.917)of T combined with DHEA-S tested by UPLC-MS/MS was not only significantly higher than the AUC(0.808)of two combined indicators tested by CLIA(P<0.001),but also significantly higher than the AUC of a single indicator--either T(P<0.01)or DHEA-S(P<0.001)tested by UPLC-MS/MS.(3)The AUC(0.942)of T,DHEA-S combined with BMI tested by UPLC-MS/MS was not only significantly higher than the AUC(0.847)of three combined indicators tested by CLIA(P<0.001),but also higher than the AUC of two combined indicators tested by UPLC-MS/MS(P<0.05).Conclusion: A reliable UPLC-MS/MS method for T,DHEA-S,AD and 17?-OHP analysis in human blood was developed and validated.T and DHEA-S tested by UPLC-MS/MS combined with BMI has a certain reference value in the diagnosis of PCOS. |
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