Measurement Of Serum Progesterone And ApoB By Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry

Objectives:To develop a candidate reference method for quantification of progesterone.by liquid chromatography tandem mass spectrometry(ID LC/MS/MS).Then the established method was used to assess the accuracy of routine immunoassays.We also evaluated the commutability of processed materials for progesterone among six routine immunoassays,with the purpose of selecting candidate reference materials for External Quality Assessment(EQA)scheme.Methods:Standard solution and related analogues were prepared to optimize mass spectrometry parameters and chromatography separation conditions of the method.In the preparation of serum samples,both liquid-liquid extraction and solid-phase extraction were investigated and the extraction recoveries were calculated.Analytical imprecision,trueness,analytical recovery,matrix effects,limits of detection(LOD),limits of quantification(LOQ)were evaluated.Then the established candidate reference method was used to assess the accuracy of routine immunoassays and commutability of processed materials.Six mainstream routine immunoassay systems domestic and foreign brand were evaluated.The processed materials were:(1)fresh frozen human serum,prepared from leftover patient serum and then pooled;(2)certified reference materials;(3)animal serum;(4)materials of EQA scheme;(5)commercial control materials;(6)commercial available sterile filtered charcoal stripped serum;(7)(2-Hydroxypropyl)-?-cyclodextrin aqueous solution.A panel of 48 fresh frozen individual samples wasmeasured with the ID-LC/MS/MS method and six immunoassays.The processed materials were tested along with the serum samples.The results of fresh frozen individual samples from ID LC/MS/MS method(x-axis)and routine method(y-axis)were analyzed by the ordinary least square and a two-tailed 95%prediction intervals(95%PIs)of the regression line were calculated.The total random error(Syx)?total analytical imprecision(Sa,tot)and biases were used to assess the routine methods.Commutability was assessed according to the 95%PIs and difference in bias approach recommend by International Federation of Clinical Chemistry(IFCC).Results:The MS used in this study was an API 5000 triple quadrupole mass spectrometer(AB Sciex,USA)coupled with an Agilent 1200 LC system(Agilent Technologies,USA).The HPLC separation was performed on a Symmetry Cl8 column(3.5?m?2.1 mm×150 mm,Waters,USA)with a mobile phase of 0.05%acetic acid in 65%acetonitrile at a flow rate of 0.25 mL/mi.Electrospray ionization(ESI)in the positive mode and multiple reaction monitoring(MRM)were used.Progesterone and progesterone-13C3 were monitored at mass transitions m/z 315.4?109.2 and m/z 318.4?112.4,respectively.The serum were first extracted by 8ml hexane and then extracted by(2-Hydroxypropyl)-?-cyclodextrin solution.Finally,progesterone was extracted into lml toluene.After evaporating,the residues were dissolved with mobile phase before analysis.The retention time of progesterone was 5.5 min.The developed method showed intra-run and total relative standard deviations(RSDs)of 0.50%?0.58%and 0.71%?1.33%,respectively.The analytical recoveries were 99.08%?101.50%.Measurement results on certified reference materials obtained with this method agreed with the certified values within the stated measurement uncertainties.The detection limit at a signal-to-noise of-3 was 1.05 pg in absolute amount.For the comparative study,the slopes of regression lines ranged from 0.961 to 1.263.The range of intercepts was-1.136?0.891 ng/mL.The six routine immunoassays had median bias between-0.199?4.06 ng/mL(-1.40%?18.85%).Sy.x was about 4-27 fold of Sa,tot at low concentrations.For the commutability based on the 95%PIs method,the fresh frozen serum pools,certified reference materials(ERM DA 347,BCR 348R GBW09197,09198,09199),commercial stripped serum were commutable among the six routine methods.EQA materials and swine serum showed positive matrix effects in some assays.(2-Hydroxypropyl)-?-cyclodextrin aqueous solution showed evident matrix effects in some methods.Based on the bias approach,none of the materials were commutable among all the routine methods.Conclusion:An ID LC/MS/MS for serum progesterone measurement was established and carefully validated.The method was precise and accurate with short analytical time.Significant calibration biases and sample-specific deviations were observed on some of the immunoassays.Noncommutability of the EQA materials was observed.HSP and CS were possible commutable EQA samples according to the 95%PIs.The results based on 95%PIs were not consistent with the IFCC's bias approach.Objectives:In order to investigate the possible strategies for calibration and procedures of preparing samples,we studied the digestion kinetics of protein and peptides,optimized the conditions for liquid chromatography mass spectrometry(LC/MS/MS)analysis,which might provide reliable research base for the development of reference measurement procedure for protein.Methods:4 specific peptides for apolipoprotein B(apo B)were selected according to the related database and other publications.Then the target peptides,isotopic labelling target peptides(used as internal standards)were synthesized.Then the conditions of LC and MS were optimized by the synthesized target peptides.Spiking with internal standard,serum were digested under different conditions(the types of trypsin,the ratio of trypsin to substrate).The samples were desalted before LC/MS/MS measurement.We recorded the absolute peak areas of target peptides and their internal standards,and calculated the peak ratios.Results:The target peptides selected for apoB were TEVIPPLIENR,TGISPLALIK,SVSLPSLDPASAK,FPEVDVLTK and there IS were TEVIPPL*IENR,TGISPL*ALIK,SVSLPSL*DPASAK,FPEVDVL*TK,respectively(*means the labelled amino acid,6 C and IN were labelled with 13C and 15N).We found that different trypsin had different digestion efficiency,thus impacted the digestion time.With specific trypsin,the samples of serum and winged-peptides could reach the utmost digestion degrees.Under 70?,the target peptides could reach the maximal peak areas and peak ratios in 30 minutes.In serum,peak areas of target peptides had the trend of decrease and the peptide of TGISPLALIK(and its internal peptide)degraded evidently and could be up to 70%in 4h.And the peak ratios of four peptides were stable in the time course of 30 min to 4 h.Conclusion:We optimized the MS and LC parameters,as well as the preparation conditions of serum samples for the quantification of apoB.By selecting appropriate trypsin and optimized reaction conditions,the digestion could be completed in 30 minutes without reducing/alkylating agents.The present results showed in our study implied that using target peptide as calibrators was possible if the complete digestion was proved,which would be helpful for accurate quantification for proteins.