| Objective To establish a method for quantification of aldoterone(ALD)in serum and urine by liquid chromatography tandem mass spectrometry method(LC-MS/MS)and compare ALD detection system based on different method.Methods This study was methodology validation research on serum and urine aldosterone evaluation using LC-MS/MS.The deuterated isotope internal was added in serum and hydrolyzed urine,followed by protein precipitation and anion exchange solid phase extraction.After SPE,the eluates were detected in the negative electro-spray ionization mode and multiple reaction monitor mode.A series of standard calibration curve were introduced for quantification of ALD.To evaluate the interference of prenisolone and cortisone which were the isomers of ALD.The precision of the method were determined by running different levels of bio-rad quality control,pooled serum and urine samples 4 times within a batch over 5days.The lower limits of quantification,recovery and stability of ALD samples were also analyzed.Urine and serum aldosterone of 80 subjects were measured by LC-MS/MS for evaluate the correlation of ALD between serum and urine.The relationship between ALD and creatinine,urea,cystatin C and eGFR were also analyzed.261 serum samples and 40 urine samples were collected and measured with LC-MS/MS and chemiluminescent immunoassays(CLIA)for ALD method comparison.193 serum samples and 113 urine samples from apparently individuals were measured with LC-MS/MS for reference interval research.Results The LC-MS/MS method established in this study can detect the concentration of serum and urine ALD simultaneously.Urine ALD was hydrolyzed at 37℃by 0.2 M hydrochloric acid for 4h.The analytical time and retention time were 4.5min and 2.2min,respectively.Linear range of ALD were good in the range of 2-1000pg/ml(R2>0.990);the repeatability and within laboratory CV of serum and urine ALD were less than 5.0%;the lower limits of quantification and lower limits of detection were 1 pg/ml and 2 pg/ml,respectively;recovery of serum and urine ALD ranged between 96.2%and 108.2%.The correlation between urine and serum aldosterone was 0.396.No correlation was found between ALD and renal function.The correlation coefficient of CLIA methods B to D and LC-MS/MS in the determination of serum ALD were 0.87,0.88 and 0.92,respectively.The mean bias of CLIA methods B to D and LC-MS/MS were-85.9 pg/ml、-72.0 pg/ml、-46.7pg/ml,respectively.The correlation coefficient of CLIA methods B,D and LC-MS/MS in the determination of urine ALD were 0.99 and 0.97.The mean bias of CLIA methods B,D and LC-MS/MS in the determination of urine ALD were-0.23 μg/24h、-1.8 μg/24h.Reference interval of serum ALD concentration(P2.5-P97.5)for LC-MS/MS method obtained from 193 apparently healthy individuals was 12.81 pg/ml to 215.99 pg/ml and reference interval of 24h urine ALD concentration(P2.5-P97.5)for LC-MS/MS method obtained from 113 apparently healthy individuals was 0.74μg/24h～15.98μg/24h.Conclusion A reliable,specific and rapid LC-MS/MS method for detecting serum and urine aldosterone simultaneously was established in clinical laboratory and can be used for clinical evaluation of ALD in human.The method was simple,rapid and it was suitable for clinical application.The consistency between CLIA and LC-MS/MS was poor. |
